Mutagenic studies that implicate specific amino acids in reaction chemistry of in substrate binding are fully interpretable only when the structural integrity of the variant protein has been validated. This validation is facilitated by identification of spin-labeled substrate analogs. Measurement of spin-probe affinity and binding stoichiometry, using a H235A and H235D mutants and comparison of these parameters with those measured for wild-type protein affords an elegant, rigorous, yet efficient solution-state method for validating the structural integrity of engineered proteins. The paramagnetic cation Mn++ is being productively used to validate the integrity of engineered variants of human HMG-CoA lyase, which catalyzed a key step in leucine catabolism. Studies were conducted using X-band EPR spectroscopy. [unreadable]